Adverse effect of superoxide-induced mitochondrial damage in granulosa cells on follicular development in mouse ovaries

Abstract

High mitochondrial oxidative phosphorylation (mt-OXPHOS) levels are required to supply the ATP necessary for follicle-stimulating hormone (FSH)-induced granulosa cell proliferation during the follicular development process. Consequently, excessive reactive oxygen species (ROS) might be generated and have an adverse effect on follicular health. This study aimed to elucidate the negative effects of ROS on mitochondrial functions in FSH-stimulated granulosa cells during the follicular development process and to investigate whether pyrroloquinoline quinone (PQQ) treatment could accelerate this process by ameliorating the adverse effects. To do this, both in vitro and in vivo experiments were performed with granulosa cells from superovulated immature (3-week-old) mice that were pretreated with or without PQQ, and a natural mating study was also performed. The ROS level in FSH-/eCG-stimulated granulosa cells was significantly increased. Moreover, high oxidative stress and mtDNA damage levels were evident in the granulosa cells. PQQ treatment not only reduced the ROS and oxidative stress levels but also ameliorated mtDNA damage, accelerated FSH-/eCG-induced ATP production, and increased the mitochondrial membrane potential and the expression levels of mitochondrial genes (Nd1, Cytb, Cox1, ATPase6) and the mt-ND1 protein. Accordingly, the proliferation and viability of granulosa cells, numbers of healthy preovulatory follicles and ovulated oocytes and serum estrogen level were significantly improved, while the apoptosis of granulosa cells was reduced. However, PQQ treatment did not change the fertility parameters in mature mice with natural cycles but did significantly increased the number of offspring born per delivery. These results revealed that ROS-associated damage in FSH-stimulated granulosa cells adversely affects their physiology and follicular health during the follicular development process. Treatment with PQQ is a beneficial tool to increase both the number of ovulated oocytes and pups per delivery.